CN103805707B - Compound amplification system and kit for detecting chromosome deficiency - Google Patents
Compound amplification system and kit for detecting chromosome deficiency Download PDFInfo
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Abstract
The invention relates to a compound amplification system and a kit for detecting chromosome deficiency. The compound amplification system comprises a mixture of a universal-specific chimeric primer pair and a universal primer, wherein the universal-specific chimeric primer pair respectively aims at genetic locus of D1S468, D1S436, D1S489, D19S112, D19S217 and D19S902. The universal primer is FluD5-Up. A sequencer of FluD5-Up is arranged at the 5' end of each forward primer in the universal-specific chimeric primer pair, and the FluD5-Up is a fluorescence labeling primer. The compound amplification system and the detection kit provided by the invention overcome the deficiency that various fluorescence labels are required for different primers, and 6 1p19q genetic locus can be detected once by using a single fluorescence label in a PCR (Polymerase Chain Reaction) system, so that the system is intuitive and reliable in result and time-saving and efficient, and can effectively detect and analyze loss of heterozigosity of the chromosome 1p19q.
Description
Technical field
The present invention relates to a kind of PCR amplification system, and use the testing product of this amplification system, belong to biological technical field.
Background technology
Being derived from neurepithelial tumour and being referred to as cerebral glioma (glioma), account for 40 ~ 50% of intracranial tumour, is modal intracranial malignant tumor, and annual morbidity is about 3 ~ 8 people/100,000 populations.Glioma is divided into 4 grades by the World Health Organization (WHO), and grade of malignancy is from minuent to height, and WHOI level is optimum, and WHOII level is low potential malignancy, and WHOIII level and WHOIV level are high malignancy.In recent years about glioma (glioma) molecular biology research of development occurs day by day deeply, the neuropathology assessment of glioma, no longer be confined to, for the clinical information providing histological type about tumour and malignant grade, the easy detection of increasing molecular marker based on histology may be needed.
Oligodendroglioma accounts for 5 ~ 18% of glioma, is a kind of separate type of glioma.There is the loss of heterozygosity (loss of heterozygosity, LOH) of No. 1 the short arm of a chromosome (1p) and No. 19 chromosome long arm (19q) in most of oligodendroglial tumors.1p and 19q loss of heterozygosity detects the early diagnosis that can be used for tumour onset.In general, the oligodendroglioma of the WHOII level of about 80%, modification oligodendroglioma between the WHOIII level of 60%, 30% ~ 50% prominent less between astrocytoma and 20% ~ 30% modification less prominent astrocytoma there is the disappearance of 1p and 19q, be less than the diffuse astrocytoma of 10% in addition, comprise in glioblastoma multiforme and carry this hereditary change.Research also finds, a kind of subclass of glioblastoma has mesoglioma source property, and this subclass and other glioblastoma are maximum in genotype is differently that 1p and 19q of this subclass has higher loss of heterozygosity rate, is respectively 40% and 60%.
1p and 19q loss of heterozygosity detects the diagnosis that also can be used for tumor recurrence, and has the patient of this molecular genetics change, and its Concurrent Chemoradiotherapy Sensitivity is higher, and prognosis is better.Caimcross(1988) and Macdonald(1990) research shows that lp/19q loss of heterozygosity (loss of heterozygosity, LOH) is the responsive important symbol of prognosis bona and PCV chemotherapy regimen (procarbazine+lomustine+vincristine(VCR)).Three perspective random III phase tests also confirm that lp/19q associating property disappearance is a strong prognostic indicator in the glioma of WHOIV level.The mesoglioma patient of research display karyomit(e) 1p/19q loss of heterozygosity is to chemosensitivity, and good prognosis, lifetime is long, the leading PCV scheme of Post operation or TMZ(Temozolomide) scheme chemotherapy that forms, radiotherapy can be postponed, as salvage treatment when recurring.
Chemotherapy can be selected after the mesoglioma excision of NCCN intracranial tumors clinical therapeutic guideline suggestion lP LOH or lp/19q LOH in 2009.In American Society of Clinical Oncology (ASCO) annual meeting in 2012, expert claims, and 1p/19q combines the tumour patient of disappearance, and combined radio chemotherapy is up-to-date standard care.In view of lp/19q disappearance accepts incontrovertible prognosis meaning in the patient of assisting therapy at oligodendroglioma, abroad 1p/19q LOH is detected as conventional sense at present, and the domestic detection just having carried out 1p/19q LOH in recent years.
At present, the method being usually used in detecting lp19q LOH comprises: fluorescence in situ hybridization (fluorescent in situ hybridization, FISH), comparative genome hybridization (comparative genomic hybridization, CGH) loss of heterozygosity and based on PCR detects (polymerasechain reaction-loss of heterozygosity, PCR-LOH), the detected result of three has good consistence.FISH only needs very little tumor sample when detecting, and do not need peripheral blood to compare, but the impact of cell section on result to be considered when analyzing, be different from hematological system tumor simultaneously, the chromosomal preparation of solid tumor and aobvious band all more difficult, need professional's operation of rich experiences.It is that the detection genome more responsive than tumour cell karyotyping obtains the method with disappearance that CGH detects, but the doping of non-tumor cell significantly can reduce the susceptibility of CGH, therefore use time need binding fiber cutting technique, and can not realize convenient, simply detect.PCR-LOH detection is the Protocols in Molecular Biology of a comparative maturity, compares with the lymphocyte of tester or healthy tissues DNA, by judging whether to there is certain disappearance with or without the appearance of am-plified fragments.Conventional determination methods comprises: Restriction Enzyme cutting method, silver dye denaturing polyacrylamide gels electrophoretic method, fluorescent mark order-checking, gel electrophoresis and quantitative PCR electrophoretic method.But because Restriction Enzyme cutting method and silver contaminates gel electrophoresis and operate quite numerous and diverse, the many factors and interpretation of result is adulterated, can not convenient, discriminating test result accurately.
In view of the foregoing, provide a kind of improvement, easily, the invention of highly sensitive karyomit(e) 1p19q loss of heterozygosity detection kit is imperative.
Summary of the invention
The technical problem to be solved in the present invention be to provide a kind of have specific nucleotide sequence general-distinctive embedment combination of primers, effectively can weaken the competition between primer and interfering factors, the composite amplification system of detected result accuracy and highly sensitive detection chromosome deletion and detection kit thereof, this amplification system can be implemented in a PCR system and adopts single fluorescent mark one-time detection 6 1p19q gene locuss.
The present invention is a kind of technical scheme solving the problems of the technologies described above proposition: a kind of composite amplification system detecting chromosome deletion, comprises the mixture of general-distinctive embedment primer pair and universal primer;
Described general-distinctive embedment primer pair is respectively for D1S468, D1S436, D1S489, D19S112, D19S217 and D19S902 gene locus; Described general-distinctive embedment primer pair in forward primer be Up-D1S468, Up-D1S436, Up-D1S489, Up-D19S112, Up-D19S217 and Up-D19S902, corresponding reverse primer is D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R;
Described universal primer is FluD5-Up, described general-distinctive embedment primer pair in each forward primer 5 ' end be provided with the sequence of FluD5-Up, described FluD5-Up is fluorescent dye primer;
Described general-nucleotide sequence of distinctive embedment primer pair and universal primer is as follows:
Prime Name Prime Sequences（5’-3’）
Up-D1S468 CTATACTGAGTCGTTGATCCGACACACACTTCCCTCTCC
D1S468-R TTAACCGTTTTGGTCCTACC
Up-D1S436 CTATACTGAGTCGTTGATCTGAATGTGTCTCCAGTGTTAGC
D1S436-R CTGTAGAGCAATCTGGCAATATGT
Up-D1S489 CTATACTGAGTCGTTGATCAGCCAGACCAAGTCTCAACA
D1S489-R CGCGTTAACATTGCTTATCAC
Up-D19S112 CTATACTGAGTCGTTGATCCAGTCATTTGAAGACCAAAGA
D19S112-R GATGGACCCACAGAAACTGA
Up-D19S217 CTATACTGAGTCGTTGATCTGAAGGGGGACCTTGAAACT
D19S217-R ATCTGGGGTTGGGGTTGATT
Up-D19S902 CTATACTGAGTCGTTGATCCCATCCTAATGAGGGCAA
D19S902-R TGCGTAGAAAATTCCCAGG
FluD5-Up CTATACTGAGTCGTTGATC。
The one of technique scheme is preferably: the concentration of above-mentioned primers F luD5-Up is 0.9 μM ~ 2.0 μMs.
Being preferably further of a kind of technique scheme: the ratio of the described concentration of primer Up-D1S468, Up-D19S217 and the concentration of primers F luD5-Up is 1: 25 to 1: 15; The ratio of the described concentration of primer Up-D1S436 and Up-D1S489 and the concentration of primers F luD5-Up is 1: 200 to 1: 150; The ratio of the described concentration of primer Up-D19S902 and the concentration of primers F luD5-Up is 1: 120 to 1: 70; The ratio of the described concentration of primer Up-D19S112 and the concentration of primers F luD5-Up is 1: 300 to 1: 250; The ratio of the described concentration of primer D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R and the concentration of primers F luD5-Up is 1: 5 to 1: 4.
Further being preferably of a kind of technique scheme: the concentration of described primers F luD5-Up is 1.8 μMs, the concentration of primer D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R is 0.4 μM, the concentration of primer Up-D1S468 and Up-D19S217 is 0.08 μM, the concentration of primer Up-D1S436, Up-D1S489 is 0.01 μM, the concentration of Up-D19S902 is 0.02 μM, and the concentration of primer Up-D19S112 is 0.007 μM.
The fluorescent mark of above-mentioned FluD5-Up is Cy5, Cy3 or FAM.
The cumulative volume of above-mentioned amplification system is 10 μ l ~ 20 μ l.
The composite amplification system of above-mentioned detection chromosome deletion also comprises the Mg of PCR damping fluid, 1.5mM ~ 2mM
2+, dNTP, 0.4U ~ the warm start enzyme of 0.6U of 0.2mM and the DNA profiling of 10ng ~ 200ng.
The present invention is the another kind of technical scheme solving the problems of the technologies described above proposition: a kind of test kit adopting the detection chromosome deletion of above-mentioned composite amplification system.
Positively effect of the present invention is:
1) multiplex PCR-CE system that the composite amplification system that the present invention detects chromosome deletion utilizes fluorescent mark universal primer to set up in conjunction with 6 pairs of Auele Specific Primers, overcome the deficiency that regular-PCR can only detect a gene locus in a reaction system, achieve detect 6 1p19q gene locuss simultaneously in a PCR system, save cost, improve efficiency.Owing to have employed specifically general-distinctive embedment primer, and apply abiotic source property universal primer and do single fluorescent mark, avoiding common multiplex PCR, need to carry out primer multiple fluorescently-labeled loaded down with trivial details, both reduced the competition between primer and interfering factors, and again saved expense.
2) concentration of the forward primer in general in amplification system of the present invention-distinctive embedment primer pair and reverse primer directly can affect the result of multiplex PCR, particularly important.Amplification system of the present invention is when preparing, make the concentration of forward primer Up-D1S468, Up-D1S436, Up-D1S489, Up-D19S112, Up-D19S217 and Up-D19S902 lower than universal primer FluD5-Up concentration, and the concentration of each primer control in suitable scope.The concentration of primers F luD5-Up preferably 1.8 μMs, the concentration of primer D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R preferably 0.4 μM, the concentration of primer Up-D1S468 and Up-D19S217 preferably 0.08 μM, the concentration of primer Up-D1S436, Up-D1S489 preferably 0.01 μM, the concentration of Up-D19S902 preferably 0.02 μM, the concentration of primer Up-D19S112 preferably 0.007 μM, this concentration proportioning can reduce competition between primer and interfering factors effectively, makes the result of multiplex PCR more reliable.
3) the capillary electrophoresis fragment analysis technology that test kit of the present invention carries out fragment analysis application is different from conventional gel electrophoretic analysis pattern, makes PCR interpretation of result more directly perceived, succinct, reliable, is easy to identify judge, is more conducive to normalizing operation.
4) simple, the simple operation of detection kit structure of the present invention, working conditions can medellings, are convenient to large-scale promotion application.
Accompanying drawing explanation
Fig. 1 is the multi-PRC reaction principle schematic of detection kit of the present invention;
Fig. 2 is the somatotype collection of illustrative plates of the blood sample of the detection kit detection sample 1 of embodiment 1;
Fig. 3 is the somatotype collection of illustrative plates of the tumor tissues DNA of the detection kit detection sample 1 of embodiment 1;
Fig. 4 is that the detection kit of embodiment 1 detects the blood sample of sample 1 and the somatotype collection of illustrative plates of tumor tissues DNA superposition;
Fig. 5 is the somatotype collection of illustrative plates of the blood sample of the detection kit detection sample 2 of embodiment 1;
Fig. 6 is the somatotype collection of illustrative plates of the tumor tissues DNA of the detection kit detection sample 2 of embodiment 1;
Fig. 7 is that the detection kit of embodiment 1 detects the blood sample of sample 2 and the somatotype collection of illustrative plates of tumor tissues DNA superposition.
Embodiment
Embodiment 1
The detection kit of the detection chromosome deletion of the present embodiment is at least made up of six test tubes that corresponding reagent is housed be fixed in paper package box, and six test tubes are amplification buffer test tube, Mg respectively
2+test tube, dNTP test tube, warm start enzyme test tube, primer mixture test tube and ultrapure water test tube.The order that the arrangement of above-mentioned test tube in packing box is placed does not specially require, and only will be clear that and indicates.
The developing principle of the detection kit of the present embodiment and using method are:
A. cerebral glioma 1p19q LOH gene locus design of primers, synthesis and screening:
Design of primers is carried out in 6 gene (D1S468, D1S436, D1S489, D19S112, D19S217, D19S902) sites according to the cerebral glioma 1p19q of international cancer association recommendation.Collect cerebral glioma person's venous whole (anticoagulated whole blood) and tissue sample (fresh tumor tissue or paraffin section), DNA cleavage liquid and Proteinase K is used to extract above-mentioned two kinds of human source gene group DNA of different nature, obtaining concentration of specimens interval is 10ng/ μ l ~ 100ng/ μ l, and high density sample is diluted with water to 100ng/ μ l; First carry out single amplification screening with above-mentioned 2 routine DNA sample to be measured, then carry out multiplex amplification, use Oligo7.0 software to carry out performance evaluation, filter out six pairs of primers.
Forward primer (Up-STR-F) 5 ' in general-distinctive embedment primer pair holds the sequence for universal primer FluD5-Up, 3 ' end is specific primer sequences section, respectively called after Up-D1S468, Up-D1S436, Up-D1S489, Up-D19S112, Up-D19S217 and Up-D19S902; Its reverse primer (STR-R) adopts 1p19q gene locus special primer, respectively called after D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R.
Universal primer adopts the primer that document has been recorded.Universal primer 5 ' end does fluorescein-labelled, uses FAM, CY3 and CY5 reporter group respectively, this universal primer called after FluD5-Up.
B.1p19q LOH gene locus multiplex PCR-CE system construction:
The PCR reaction system that the test kit of the present embodiment adopts is multi-PRC reaction system, and namely 6 gene locus special primers and fluorescent mark universal primer are placed in same PCR pipe and increase.10 μ l ~ 20 μ l systems are adopted all can better to increase.In consideration cost factor and after PCR experiment repeatedly adjusts and optimizes, preferred multi-PRC reaction system cumulative volume is 10 μ l, comprises the Mg of 10ng ~ 200ng genomic dna, PCR damping fluid, 2mM
2+, 0.2mM the warm start enzyme of dNTP, 0.5U, and primers F luD5-Up 1.8 μMs, each 0.4 μM of D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R, D19S902-R, each 0.08 μM of Up-D1S468, Up-D19S217, each 0.01 μM of Up-D1S436, Up-D1S489, Up-D19S902 0.02M, Up-D19S112 0.007 μM.
In multi-PRC reaction system, the concentration of the forward primer Up-STR-F in general-distinctive embedment primer pair is number/mono-of universal primer FluD5-Up concentration, and its concentration adjusts because of the difference of each gene PCR amplification efficiency.Because Up-STR-F has the gene order of 1p19q site complementation, therefore in PCR reaction process, first primer Up-STR-F and STR-R carry out the amplification of 5 ~ 10 circulations under the effect of warm start enzyme to 1p19q gene, produces the complementary fragment that 5 ' end band has Up.After Up-STR-F exhausts, FluD5-Up and STR-R carries out following amplification to above-mentioned pcr amplification product under the effect of warm start enzyme, and producing 5 ' end band has fluorescently-labeled gene fragment complementary.The principle of above-mentioned PCR reaction as shown in Figure 1.
The preferred PCR reaction conditions of detection kit of the present embodiment is: 95 DEG C of preheatings 5 minutes; Then 94 DEG C of sex change 10 seconds, 54 DEG C of annealing 10 seconds, 72 DEG C extend 30 seconds, totally 40 circulations; Last 72 DEG C extend 5 minutes, 4 DEG C of insulations.
PCR primer through above-mentioned steps gained carries out capillary electrophoresis (capillary electrophoresis, CE).Utilize multiple fluorescence PCR to detect 1p19q gene locus in conjunction with capillary electrophoresis technique, there is the advantages such as efficient, stable, highly sensitive, analytical results is reliable.Preferred capillary electrophoresis scheme is: each sample CE system cumulative volume is 20 μ l, comprise above-mentioned PCR primer 1 μ l ~ 5 μ l, DNA-Size reference liquid 0.2 μ l ~ 0.5 μ l, and sample-loading buffer (Sample Loading Solution, SLS), finally cover 1 dropstone wax oil.Electrophoresis gained fluorescence pattern carries out fragment analysis.
C. the analysis of cerebral glioma 1p19q LOH gene fragment judges:
Whole blood and tumor tissues specimen dna are carried out capillary electrophoresis after amplification, and gained collection of illustrative plates is compared.Compare according to the gene fragment size indicated in collection of illustrative plates and photoluminescence peak peak shape, if compared with whole blood sample, tumor tissues occurs that namely a certain gene band fluorescent signal minimizing more than 50% or band disappearance are judged as 1p or 19q loss of heterozygosity.
The use step of the detection kit of the present embodiment is: the DNA extraction of first carrying out Patients with gliomas tumor tissues and contrast; Apply multiplex amplification system again to increase; Last amplified production gained collection of illustrative plates after capillary electrophoresis carries out 1p19q LOH and analyzes judgement.
Below the concrete performance of two routine Patients with gliomas being carried out to 1p/19q LOH detection:
1, whole blood and tumor tissues paraffin section DNA extraction:
These 1 or 2 whole blood sample 400 μ l of sampling, add 1ml distilled water, latter centrifugal 5 minutes of mixing, discard upper liquid 1ml, add 400 μ l lysates and 20 μ l Proteinase Ks, 70 DEG C of metal baths 10 minutes, cross centrifugal column, after 500 μ l scavenging solutions successively cleaning twice, add the centrifugal wash-out of 50 μ l elutriant, survey sample DNA concentration value.
This tumor tissues paraffin section 5 of 1 or 2 of sampling, scraping blade enters 1.5ml centrifuge tube, add the dewaxing of 1ml dimethylbenzene, after 1ml ethanol purge twice, add 400 μ l lysates and 20 μ l Proteinase Ks, 60 DEG C of metal baths 16 hours, cross centrifugal column, after 500 μ l scavenging solutions successively cleaning twice, add the centrifugal wash-out of 50 μ l elutriant, survey sample DNA concentration value.
2,1p/19q LOH detects, and concrete operation step is as follows:
A. 10 × PCR damping fluid 1 μ l, 100mM Mg is added in each reaction system
2+0.2 μ l, 2.5mM dNTP 0.8 μ l, 5U warm start enzyme 0.1 μ l; Up-D1S468, Up-D1S436, Up-D1S489, Up-D19S112, Up-D19S217 and Up-D19S902 of 10 μMs respectively get 0.4 μ l by after the Dilution ratio dilution of 1:5,1:40,1:40,1:60,1:5,1:20 respectively; D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R of 10 μMs respectively get 0.4 μ l; The FluD5-Up of 50 μMs gets 0.36 μ l; Above-mentioned DNA profiling 2 μ l; Last moisturizing to 10 μ l.
Adopt the sequence of primer following (5 '-3 '):
D1S468 primer
Up-D1S468 CTATACTGAGTCGTTGATCCGACACACACTTCCCTCTCC
D1S468-R TTAACCGTTTTGGTCCTACC
D1S436 primer
Up-D1S436 CTATACTGAGTCGTTGATCTGAATGTGTCTCCAGTGTTAGC
D1S436-R CTGTAGAGCAATCTGGCAATATGT
D1S489 primer
Up-D1S489 CTATACTGAGTCGTTGATCAGCCAGACCAAGTCTCAACA
D1S489-R CGCGTTAACATTGCTTATCAC
D19S112 primer
Up-D19S112 CTATACTGAGTCGTTGATCCAGTCATTTGAAGACCAAAGA
D19S112-R GATGGACCCACAGAAACTGA
D19S217 primer
Up-D19S217 CTATACTGAGTCGTTGATCTGAAGGGGGACCTTGAAACT
D19S217-R ATCTGGGGTTGGGGTTGATT
D19S902 primer
Up-D19S902 CTATACTGAGTCGTTGATCCCATCCTAATGAGGGCAA
D19S902-R TGCGTAGAAAATTCCCAGG
Be with fluorescently-labeled universal primer
FluD5-Up CTATACTGAGTCGTTGATC。
B. be placed on amplification instrument by PCR reaction tubes, the PCR response procedures of operation is: 95 DEG C of preheatings 5 minutes; Then 94 DEG C of sex change 10 seconds, 54 DEG C of annealing 10 seconds, 72 DEG C extend 30 seconds, totally 40 circulations; Last 72 DEG C extend 5 minutes.4 DEG C of preservations.
C. gained PCR primer is placed on Beckman GenomelabGeXP genetic analyzer, capillary electrophoresis is carried out by instrument operation instruction, each CE system cumulative volume is 20 μ l, comprise above-mentioned PCR primer 1 μ l, DNA-Size reference liquid 0.5 μ l, Sample Loading Solution 18.5 μ l, finally cover 1 dropstone wax oil.CE gained fluorescence pattern carries out gene fragment analysis.
3, cerebral glioma 1p19q gene fragment is analyzed:
The whole blood of above-mentioned sample 1 or 2 and tumor tissues sample CE collection of illustrative plates are carried out gene fragment analysis, synchronous compare is made according to the gene fragment size indicated in collection of illustrative plates and photoluminescence peak peak shape, as shown in Figures 2 to 7, X-coordinate is gene fragment base numerical value, ordinate zou is fluorescence values, X-coordinate gene fragment order is from left to right followed successively by for D19S112, D1S489, D1S468, D1S436, D19S217, the amplified fragments of D19S902, wherein Fig. 2 and Fig. 5 is whole blood sample gene fragment analysis chart, Fig. 3 and Fig. 6 is tumor tissues sample chips piecewise analysis figure, Fig. 4 and Fig. 7 is the comparison diagram after superposition.Wherein all there is not the deficient phenomena of gene fragment in sample 1, therefore be judged to be that 1p and 19q does not lack; There is band deficient phenomena, so be judged to be 19q LOH in the tumor tissues sample D19S902 gene fragment of sample 2.
To blood sample and the tumor tissues sample of sample 1 and 2, adopt respectively: (1) D1S468 primer+universal primer; (2) D1S436 primer+universal primer; (3) D1S489 primer+universal primer; (4) D19S112 primer+universal primer; (5) D19S217 primer+universal primer; (6) D19S902 primer+universal primer, adopts capillary electrophoresis analysis after carrying out substance pcr amplification, and result of determination is consistent with above-mentioned multiplex PCR result.
Embodiment 2
The rest part of the multiplexed detection reagents box of the detection chromosome deletion of the present embodiment is identical with embodiment 1, and difference is: the forward primer in general-distinctive embedment primer pair is different with the concentration of reverse primer.
Embodiment 3
The rest part of the multiplexed detection reagents box of the detection chromosome deletion of the present embodiment is identical with embodiment 1, and difference is: the forward primer in general-distinctive embedment primer pair is different with the concentration of reverse primer.
Comparative example 1 to comparative example 3
The rest part of the detection kit of comparative example 1 to comparative example 3 is identical with embodiment 1, and difference is: the forward primer in general-distinctive embedment primer pair is different with the concentration of reverse primer.
Forward primer in the detection kit of embodiment 1 to 3 and comparative example 1 to 3 and the concentration of reverse primer as shown in table 1:
Table 1 primer concentration table
The detected result of the detection kit of embodiment 2 and 3 is consistent with the detected result of embodiment 1, the detected result of the detection kit of comparative example 1 to 3 and the detected result deviation to some extent of embodiment 1, as shown in table 2, it can thus be appreciated that, amplification system is when preparing, make the concentration of forward primer Up-D1S468, Up-D1S436, Up-D1S489, Up-D19S112, Up-D19S217, Up-D19S902 lower than universal primer FluD5-Up concentration, otherwise fluorescent signal can be made too weak; And the concentration of each primer should control in suitable scope, otherwise the interference between primer can be caused, when some tissue samples concentration is on the low side, more easily make the amplification efficiency in some site too low and cannot identification, cause judging.
Embodiment 1 to embodiment 3 makes to reach balance between the peak type of each site amplified fragments by optimizing primer concentration.If different tissues gene amplification efficiency is suitable, electrophorogram can be superimposed to analyze, as Fig. 4 and Fig. 7; If wherein a certain tissue augmentation efficiency is lower, can the some multiples of corresponding amplification to analyze, magnification with the clear and legible knowledge of object fragment for standard.
Table 2 detected result table
Obviously, above-described embodiment is only for example of the present invention is clearly described, and is not the restriction to embodiments of the present invention.For those of ordinary skill in the field, can also make other changes in different forms on the basis of the above description.Here exhaustive without the need to also giving all embodiments.And these belong to spirit institute's apparent change of extending out of the present invention or change and are still among protection scope of the present invention.
Claims (6)
1. detect a composite amplification system for chromosome deletion, it is characterized in that: the mixture comprising general-distinctive embedment primer pair and universal primer;
Described general-distinctive embedment primer pair is respectively for D1S468, D1S436, D1S489, D19S112, D19S217 and D19S902 gene locus; Described general-distinctive embedment primer pair in forward primer be Up-D1S468, Up-D1S436, Up-D1S489, Up-D19S112, Up-D19S217 and Up-D19S902, corresponding reverse primer is D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R;
Described universal primer is FluD5-Up, and described FluD5-Up is fluorescent dye primer;
Described general-nucleotide sequence of distinctive embedment primer pair and universal primer is as follows:
Prime Name Prime Sequences（5’-3’）
Up-D1S468 CTATACTGAGTCGTTGATCCGACACACACTTCCCTCTCC
D1S468-R TTAACCGTTTTGGTCCTACC
Up-D1S436 CTATACTGAGTCGTTGATCTGAATGTGTCTCCAGTGTTAGC
D1S436-R CTGTAGAGCAATCTGGCAATATGT
Up-D1S489 CTATACTGAGTCGTTGATCAGCCAGACCAAGTCTCAACA
D1S489-R CGCGTTAACATTGCTTATCAC
Up-D19S112 CTATACTGAGTCGTTGATCCAGTCATTTGAAGACCAAAGA
D19S112-R GATGGACCCACAGAAACTGA
Up-D19S217 CTATACTGAGTCGTTGATCTGAAGGGGGACCTTGAAACT
D19S217-R ATCTGGGGTTGGGGTTGATT
Up-D19S902 CTATACTGAGTCGTTGATCCCATCCTAATGAGGGCAA
D19S902-R TGCGTAGAAAATTCCCAGG
FluD5-Up CTATACTGAGTCGTTGATC；
The concentration of described primers F luD5-Up is 0.9 μM ~ 2.0 μMs;
The ratio of the described concentration of primer Up-D1S468, Up-D19S217 and the concentration of primers F luD5-Up is 1: 25 to 1: 15; The ratio of the described concentration of primer Up-D1S436 and Up-D1S489 and the concentration of primers F luD5-Up is 1: 200 to 1: 150; The ratio of the described concentration of primer Up-D19S902 and the concentration of primers F luD5-Up is 1: 120 to 1: 70; The ratio of the described concentration of primer Up-D19S112 and the concentration of primers F luD5-Up is 1: 300 to 1: 250; The ratio of the described concentration of primer D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R and the concentration of primers F luD5-Up is 1: 5 to 1: 4.
2. the composite amplification system of detection chromosome deletion according to claim 1, it is characterized in that: the concentration of described primers F luD5-Up is 1.8 μMs, the concentration of primer D1S468-R, D1S436-R, D1S489-R, D19S112-R, D19S217-R and D19S902-R is 0.4 μM, the concentration of primer Up-D1S468 and Up-D19S217 is 0.08 μM, the concentration of primer Up-D1S436 and Up-D1S489 is 0.01 μM, the concentration of Up-D19S902 is 0.02 μM, and the concentration of primer Up-D19S112 is 0.007 μM.
3. the composite amplification system of detection chromosome deletion according to claim 1 and 2, is characterized in that: the fluorescent mark of described FluD5-Up is Cy5, Cy3 or FAM.
4. the composite amplification system of detection chromosome deletion according to claim 1 and 2, is characterized in that: the cumulative volume of described amplification system is 10 μ l ~ 20 μ l.
5. the composite amplification system of detection chromosome deletion according to claim 1 and 2, is characterized in that: the Mg also comprising PCR damping fluid, 1.5mM ~ 2mM
2+, dNTP, 0.4U ~ the warm start enzyme of 0.6U of 0.2mM and the DNA profiling of 10ng ~ 200ng.
6. one kind adopts the test kit of the detection chromosome deletion of composite amplification system as claimed in claim 1.
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| CN110106063B (en) * | 2019-05-06 | 2022-07-08 | 臻和精准医学检验实验室无锡有限公司 | System for detecting 1p/19q combined deletion of glioma based on second-generation sequencing |
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